The gut-associated lymphoid tissue (GALT) is critical for the initial establishment and spread of HIV-1 infection. Nearly all transmitted/founder HIV-1 strains utilize CCR5 as a coreceptor, and large numbers of activated effector memory CD4+ T cells expressing CCR5 are found in the GALT. It is currently thought that host innate immune responses dictated type I IFNs (IFNa and IFNB play key roles in counteracting initial viral spread. IFNa/B are induced in early HIV-1 infection, and plasmacytoid dendritic cells (pDCs), which produce IFNa/B in large quantities, are rapidly recruited to mucosal sites of SIV infection. IFNa/B likely inhibits HIV-1 by increasing the expression and/or activity of restriction factors,or proteins that could directly inhibit HIV-1 replication. Over 30 HIV-1 restriction factors have been identified including APOBEC3G, Tetherin, Mx2 and TRIM5a. However, our studies in a mou e model of retrovirus infection revealed that single IFN-stimulated genes (ISGs) could dominantly account for the antiretroviral potency of IFNa1. Notably, IFNa/B and pDC activation were also linked to chronic immune activation, a significant predictor of HIV-1 disease progression. These events could be driven by early Th17 cell depletion and epithelial barrier dysfunction leading to the translocation of microbial products into the lamina propria. The mechanism(s) underlying the double-edged impact of IFNa remains unclear, but IFNa is comprised of 12 distinct subtypes that possess diverse biological properties2. However, the interplay between type I IFNs, R5-tropic HIV-1, mucosal lymphocytes, commensal enteric bacteria and restriction factors remains a major knowledge gap in the field. To this end, we developed the Lamina Propria Aggregate Culture (LPAC) model to quantify gut CD4+ T cell death by R5-tropic HIV-1 in the context of microbiota3. Here, we propose to utilize this innovative LPAC model to answer: 1. Which IFNa subtypes are induced during acute versus chronic HIV-1 infection in the GALT? 2. How do type I IFNs impact mucosal HIV-1 infection, Th/DC activation and T cell depletion? 3. Which restriction factors serve as effectors of type I IFNs in mucosal CD4+ T cells? To answer these questions, we assembled a unique collaborative team with expertise in HIV-1 virology and restriction factors, mucosal immunopathogenesis and IFNa subtypes. Our research team will employ next- generation sequencing approaches to quantify IFNa subtype distribution in primary pDCs and colon pinch biopsies from HIV-infected and HIV-uninfected individuals. We will produce recombinant IFNa subtypes and hybrids to map IFNa residues involved in antiretroviral versus immunomodulatory activity. We will utilize gene knock-out technologies to dissect the role of restriction factors in type I IFN-mediated HIV-1 inhibition. The results should provide urgently needed insights on how type I IFNs may impact mucosal HIV-1 infection and pathogenesis and inform strategies that aim to either harness IFNa for antiretroviral therapies or block type I IFN-mediated immune activation to reduce chronic inflammation in the GALT.